| Three genetic systems co-exit in the plant cells which are nuclear genome, chloroplast genome and mitochondrial genome respectively. DNAs flow among the three genomes occur frequently which can be proved by comparing the mitochondrial sequences with the genomic sequences of model plant rice and Arobidopsis. Gene transfer from the mitochondrion to the nucleus, a process of outstanding importance to the evolution of the eukaryotic cell, is an on-going phenomenon in higher plants. After transfer, the mitochondrial gene has to be adapted to the nuclear context by acquiring a new promoter and targeting information to direct the protein back to the organelle. Various gene transfers from mitochondrion to nuclear genome had been discovered after completing the whole genome sequencing of various model species. However, it is difficult to sequence the whole genome of wheat for its large genome and limited number of BAC clones sequenced so far. Consequently, the study on mitochondrial genes transferring to nucleus at the whole genome level has not been reported.Fifty-one BAC clones were sequenced which derived from the Sr2/FHB region of wheat chromosome 3B in this paper. The total sequence length is about 43Mb with the average reads >700bp and the average coverage >5×. A continuous 52kb mitochondrial DNA insertion was disvovered with similarity >98% by sequence comparison with mitochondrial genome sequence. To better understand the process of the mitochondrial genes insertion event, we investigated the insertion and integration mechanisms and evolution of the 52kb mitochondrial sequence locus in wheat genome. The results are as follows:The ~52kb nuclear mitochondrion (NUMT) was located on chromosome 3B by PCR analysis of Chinese Spring nulli-tetrasomics. PCR amplification of di-, tetra- and hexaploid materials using genome-specific primers indicated that the NUMT inserted in the nucleus when the tetraploid wheat speciated or after that time and retained until the hexaploid wheat speciated. The NUMT derived from rrn18-1/nad1a and rrn18-2/trnI regions of mitochondrion. Due to recombination taking place, three separate NUMTs were shaped with different directions with mitochondrion in the nucleus and designated as 38 Tma 3 B, 12 Tma 3 Band 2 Tma 3 B respectively. This NUMT accounted for 11.5% of the whole mitochondrial genome and contained 12 mitochondrial genes and 4 repetitive regions- R2 R4 R14 and R15. There was a phenomenon that genes exist in the repeats such as trnP-1 was in R14, trnD-1 was in R15 and rrn5-1 and rrn18-1 were in R4 while R4 was in R2. Six genes nad7,rrn5, rrn18,trnfM,rps19-P and nad4L were supported by ESTs of wheat and/or rice at different levels by searching the GenBank database and the full length cDNA sequence database in our lab which implied that the six genes express in the nuclear genome. Synteny between the wheat NUMT and rice genome exited: there was a two-gene cluster(trnN-nad1)on rice chromosome 1, a two-gene cluster(nad7c-rrn18)on chromosome 3, a two-gene cluster(trnS-rrn18)on chromosome 4, a two-gene cluster(rrn18-rrn5)on chromosome 7 and two two-gene clusters(rps2-trnS and rrn18-rrn5) on chromosome 12. InDels and nucleotide substitutions were found by sequence pair-wise comparison. The largest deletion is 68 nucleotides, and the largest insertion is 10 nucleotides; the rate of substitution of 38 Tma 3 B,12 Tma 3 B and 2 Tma 3 B are 7.71, 8.20 and 7.26 nt/kb respectively. The transition frequency is higher than transversion, C→T and G→A transitions formed the highest-frequency substitution class. The evolution time of the insertion was dated which indicates that the insertion fragment which derived from different location of mitochondria transferred to nucleus at the same time. The experimental data support the random end-joining hypothesis. Analysis of sequences flanking the insertion showed that retrotransposons and transposons (class 1 and class 2 elements) are predominant repeats which formed 6 repeat regions while SSRs are abundant in this region. Eighteen genes are predicted and the gene density is one gene per 14kb. CDS14 was identified as EXPB11 gene. Experimental data indicated that the large continuous insert located in the region with low level of repeats (repeats proportion is 35% lower than the average level of 80%) and with high level of gene density. |
